Urine HPV in the Context of Genital and Cervical Cancer Screening—An Update of Current Literature

Within the previous decades, following the widespread implementation of HPV-related biomarkers and computerization in liquid-based cytology, screening for lower genital tract malignancies has been optimized in several parts of the world. Many organized anogenital cancer prevention systems have reached a point at which efficacy is more a matter of population coverage and less of available infrastructures. Meanwhile, self-sampling modalities in which biologic material (vaginal secretions, urine, etc.) is obtained by the individual and not the clinician and subsequently undergoes examination for HPV biomarkers enjoy appreciating acceptance. Bygone the initial skepticism that vaginal or urine HPV represents “passenger” transient infections, extensive scientific work has been conducted to optimize high-risk HPV (hrHPV) detection from this “novel” biologic material. Nowadays, several state-of-the-art meta-analyses have illustrated that self-sampling techniques involving urine self-sampling represent a feasible alternative strategy with potentially enhanced population coverage possessing excellent performance and sensitivity. Recently published scientific work focusing on urine HPV was reviewed, and after a critical appraisal, the following points should be considered in the clinical application of hrHPV urine measurements; (i) use of first-void urine (FVU) and purpose-designed collection devices; (ii) using a preservation medium to avoid human/HPV DNA degradation during extraction and storage; (iii) using polymerase chain reaction (PCR) based assays, ideally with genotyping capabilities; (iv) processing of a sufficient volume of whole urine; and (v) the use of an analytically sensitive HPV test/recovery of cell-free HPV DNA in addition to cell-associated DNA.

Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles

BackgroundUrinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear.MethodsUrine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization.ResultsUrine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47–0.95) and patients (R2 0.41–0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers.Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment–specific EVs.

Increased urinary exosomal SYT17 levels in chronic active antibody-mediated rejection after kidney transplantation via the IL-6 amplifier

Chronic active antibody-mediated rejection (CAAMR) is a particular problem in kidney transplantation (KTx), and ~25% of grafts are lost by CAAMR. Further, the pathogenesis remains unclear, and there is no effective cure or marker. We previously found that a hyper NFκB-activating mechanism in non-immune cells, called the IL-6 amplifier, is induced by the co-activation of NFκB and STAT3, and that this activation can develop various chronic inflammatory diseases. Here, we show that synaptotagmin-17 (SYT17) is increased in an exosomal fraction of the urine from CAAMR patients, and that this increase is associated with activation of the IL-6 amplifier. Immunohistochemistry showed that SYT17 protein expression was increased in renal tubule cells of the CAAMR group. While SYT17 protein was not detectable in whole-urine samples by western blotting, urinary exosomal SYT17 levels were significantly elevated in the CAAMR group compared to three other histology groups (normal, interstitial fibrosis and tubular atrophy, and calcineurin inhibitors toxicity) after KTx. On the other hand, current clinical laboratory data could not differentiate the CAAMR group from these groups. These data suggest that urinary exosomal SYT17 is a potential diagnostic marker for CAAMR.

The Urinary Transcriptome as a Source of Biomarkers for Prostate Cancer

Prostate cancer (PCa) is the second most common cancer of men and is typically slow-growing and asymptomatic. The use of blood PSA as a screening method has greatly improved PCa diagnosis, but high levels of false positives has raised much interest in alternative biomarkers. We used next-generation sequencing (NGS) to elucidate the urinary transcriptome of whole urine collected from high-stage and low-stage PCa patients as well as from patients with the confounding diagnosis of benign hyperplasia (BPH). We identified and validated five differentially expressed protein-coding genes (FTH1 BRPF1, OSBP, PHC3, and UACA) in an independent validation cohort of small-volume (1 mL) centrifuged urine (n = 94) and non-centrifuged urine (n = 84) by droplet digital (dd)PCR. These biomarkers were able to discriminate between BPH and PCa patients and healthy controls using either centrifuged or non-centrifuged whole urine samples, suggesting that the urinary transcriptome is a valuable source of non-invasive biomarkers for PCa that warrants further investigation.

Influence of preanalytic and analytic variables in canine and feline urine specific gravity measurement by refractometer

Urine specific gravity (USG), which is usually measured by refractometry, is an important indicator of renal concentrating ability. Few studies have evaluated refractometers with separate scales for canine and feline urine. Variables such as protein content or storage time may influence the USG. We compared the effects of measuring USG with a refractometer with single or separate scales for canine and feline urine, investigated inter- and intra-observer variability, and measured agreement between whole urine and supernatant. We evaluated the correlation between USG and osmolality, the influence of urinary protein on USG and osmolality, and the impact of storage time up to 6 mo. We examined 252 canine and 126 feline samples. Bland–Altman analysis revealed higher USG values of the single-scale refractometer than the dual-scale refractometer, with a mean difference (bias) of < 0.001 for canine and 0.003 for feline specimens. Inter- and intra-observer variability were acceptable. Good agreement was shown between USG of whole urine and supernatant. Correlations between USG and osmolality were excellent (0.98–0.99, p < 0.001). Proteinuria up to 1 g/L had no major impact on USG or osmolality. Storage time had no significant effect on USG. The difference between the refractometers is clinically irrelevant, and the use of a refractometer with separate feline and canine scales is unnecessary. Whole urine and supernatant stored up to 6 mo can both be used for USG measurement. The influence of proteinuria <1 g/L on USG and osmolality is negligible.

Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine

ABSTRACT Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold (CT) was used to measure amplifiable cfDNA. In spiked samples, the median CT values for M. tuberculosis, S. enterica, and EBV cfDNA were significantly lower in blood collected in K2EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen CT values for Streck and PAXgene but not K2EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at –80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest that large-volume single-spin K2EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.